Design, engineering, and production of human recombinant T cell receptor ligands derived from human leukocyte antigen DR2

Major histocompatibility complex (MHC) class II molecules are membrane-anch ored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antig en-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, incl uding human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1 *1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formatio n of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*010 1) and HLA-DR2b (DRA*0101(al, DRB1*1501). Both isotypes can present an immu nodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated th at the peptide binding/T cell recognition domains of rat MHC class II (alph a1 and pi domains) could be expressed as a single exon for structural and f unctional characterization; Burrows, G. G., Chang, J. m,, Bachinger, H.-P., Bourdette, D. N., Wegmann, K, W, Offner H, and Vandenbark A. A. (1999) Pro tein Eng. 12, 711-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Cha ng, J. W., Tenditnyy, K., Vandenbark, A. A, and Offner, H. (2000) J. Immuno l 164, 6366-6371). Single-chain human recombinant T cell receptor ligands ( RTLs) of similar to 200 amino acid residues derived from HLA-DR2b were desi gned using the same principles and have been produced in Escherichia coil w ith and without amino-terminal extensions containing antigenic peptides. St ructural characterization using circular dichroism predicted that these mol ecules retained the antiparallel beta -sheet platform and antiparallel alph a -helices observed in the native HLA-DR2 heterodimer. The proteins exhibit ed a cooperative two-state thermal unfolding transition, and DR2-derived RT Ls with a covalently linked MBP peptide (MBP-(85-99)) showed increased stab ility to thermal unfolding relative to the empty DR2-derived RTLs. These no vel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing p otent and selective human diagnostic and therapeutic agents for treatment o f autoimmune disease.